An RNA-hydrolyzing recombinant minibody prevents both influenza A virus and coronavirus in co-infection models … – Nature.com

Cell lines

Madin-Darby canine kidney (MDCK) (ATCC CCL-34) cells were maintained in complete Eagles minimal essential medium (MEM) (Hyclone, USA). Vero E6 cells (ATCC CRL-1586) were grown in complete Dulbeccos modified Eagles medium (DMEM) (Hyclone, USA) at 37C and 5% CO2. The complete media were supplemented with 10% fetal bovine serum (Hyclone, USA) and 1% antibioticantimycotic (ThermoFisher Scientific, USA).

IAV strains A/Puerto Rico/8/1934 H1N1 and H3N2/A/X-31, which were kindly provided by Prof. Dae-Hyuk Kweon (Sungkyunkwan University, Republic of Korea), were propagated in the allantoic fluid of 9-day-old embryonated chicken eggs at 37C. The viruses were then collected and purified using sucrose gradient centrifugation. Vero E6 cell-adapted H1N1 (adapted-H1N1) was successfully generated from H1N1/PR8 as parental virus via virus adaptation method and propagated in Vero E6 cells. hCoV-OC43 (KBPV-VR-8) and PEDV (CV777), which were kindly provided by Novelgen research center (Suwon, Republic of Korea), were propagated in Vero E6 cells. All viral titers were determined by plaque assays.

3D8 scFv gene was constructed in pIg20-3D8 plasmid (Fig.S1a), and expressed via the addition of 1mM isopropyl 1-thiol-D-galactopyranoside (IPTG) to Escherichia coli BL21(DE3) pLysE strain in LuriaBertani broth enriched with 100g/mL ampicillin and 25g/mL chloramphenicol for 18h at 26C. The cell culture supernatant was obtained using centrifugation at 6000rpm for 20min at 4C and then filtered through a 0.22-mm filter. Next, 3D8 scFv was purified from the supernatant using an IgG Sepharose 6 fast-flow affinity column (GE Healthcare, USA). 3D8 scFv was then eluted with acetic acid (0.1M, pH 3.4), and neutralized with a 0.1 volume of 1M TrisHCl (pH 9.0). The purity of the eluted protein was confirmed by SDS-PAGE with Coomassie-blue staining. All its key features were checked before being used for further experiments (Fig.S1b, c and d).

In order to confirm nucleic acid-hydrolyzing activity of 3D8 scFv, several types of substrates were used including pUC19 vector, ribosomal RNA, and viral RNA as double-stranded DNA, RNA substrates, respectively. The substrates (1g) were incubated with 0.5g of 3D8 scFv in 1X tris-buffered saline containing 0.1mM MgCl2 at 37C for 0-15-30-0min, then analyzed using electrophoresis on a 1% agarose gel and stained with ethidium bromide. Total RNA was extracted from cell lines by using TRI reagent (MRC, USA), and viral RNA was synthesized using HiScribe T7 high yield RNA synthesis kit (New England Biolabs, USA) according to the manufacturing protocol.

Three models of co-infection were established: (1) two coronaviruses [hCoV-OC43 (MOI 0.1) and PEDV (MOI 0.02)], (2) two IAVs [H1N1/PR8 (MOI 1) and H3N2/X-31(MOI 2)], and (3) coronavirus and influenza virus [hCoV-OC43 [MOI 0.1 and adapted-H1N1 (MOI 1)]. For model (2), the two IAVs were inoculated simultaneously to MDCK cells (1105 cells/well) seeded on a 24-well plate (SPL Life Sciences, Republic of Korea) for 1h. After that, the infection medium was removed and serum-free medium containing 0.2% BSA and 1g/mL TPCK was then added to the virus-infected cells. At 6h post-infection (hpi), 10M of 3D8 scFv was added to the cells, followed by incubation at 37C, 5% CO2 for 24h. For model (1) and (3), Vero E6 cells (2105 cells/well) were seeded on a 24-well plate, washed twice with Dulbecco's phosphate-buffered saline (DPBS), and challenged with different pairs of viruses in serum-free media. Following 1h of absorption, the infection medium was removed and replaced with complete DMEM for model (1) and DMEM including 0.2% BSA and 1g/mL TPCK for model (3). At 6 hpi, the cells were treated with 3D8 scFv. After that, supernatants and cells were collected at 54 hpi for model (1) and at 30 hpi for model (2) and (3), followed by storage at80C for further experiments. In which, cells were harvested using TRI reagent (MRC, USA) and RIPA buffer (Santa Cruz Biotechnology, USA) for RNA and protein extraction, respectively.

Total RNA was isolated using TRI reagent (MRC, USA), a final RNA concentration of 10ng/L was used. One-step RT-qPCR was performed using AccuPower GreenStar RT-qPCR Premix and Master mix (Bioneer, Republic of Korea) and Rotor-Gene Q system (Qiagen, German) with 50ng of RNA template. Influenza virus genes (HA and NA), coronavirus genes (N and S), a toll-like receptor gene (TLR-7) were amplified using the primers listed in Table S1.

MDCK and Vero E6 cells were seeded at 7105 cells/well in 6-well plates to 90100% confluency. The supernatants harvested in the antiviral tests were serially diluted tenfold, and 1mL of the diluted viral suspension was inoculated to DPBS-washed cells. Following 1h of incubation, the cells were overlaid with DMEM containing 1% SeaPlaque agarose (Lonza, USA) with 1g/mL TPCK for model (2) and (3) or without TPCK for model (1). Plaque formation was observed for 3 and 4 days of incubation for influenza virus and coronavirus, respectively. Plaques were counted, and the percentage of plaque reduction was calculated.

Cells were lysed using RIPA buffer (Santa Cruz Biotechnology, USA) to extract the protein. Next, 20g of the protein was subjected to SDS-PAGE. Membranes after being transferred from gels were incubated with primary antibodiesmonoclonal antibody to PEDV nucleoprotein protein (clone 3F12, 9191, Median Diagnostics, Republic of Korea), monoclonal antibody to hCoV-OC43 nucleoprotein (clone 542-7D, LS-C79764, LS-bio, USA), polyclonal rabbit anti-HA antibody to IAV (including H1N1 and H3N2) (PA5-349291, Invitrogen, USA), and polyclonal rabbit anti-GAPDH antibody (ab9485, Abcam, UK). After that, membranes were incubated with goat anti-mouse IgG-HRP conjugate (G-21040 Invitrogen, USA), and goat anti-rabbit IgG-HRP conjugate (A21020, Abbkine, USA). The membranes were added with Enhanced chemiluminescence (W3652-050, DawinBio, Republic of Korea) and exposed the film to observe the results. To analyze samples of viral co-infection, samples were divided into separated sets and then SDS-PAGEs were performed and transferred onto separated membranes. Each membrane was treated with different primary antibodies and secondary antibodies.

Vero E6 cells (2104) were cultured in 8-well chamber slides. The hCoV-OC43 and adapted-H1N1 co-infection and 3D8 scFv treatment were performed as described above. The slides were fixed with cold-methanol and permeabilized with an Intracellular Staining Perm Wash Buffer (Biolegend, USA) for 15min each. Following blocking with PBS with 0.1% tween 20 containing 1% BSA and glycine for 1h, the cells were incubated with polyclonal rabbit anti-HA antibody (PA5-349291, Invitrogen, USA), monoclonal anti-coronavirus antibody (OC43 strain, clone 541-8F, MAB9012, Sigma-Aldrich, USA), and anti-3D8 antibody (humanized antibody, clone 1D7, Bioneer, Republic of Korea) at 1:1000 dilution for 24h at 4C. After that, goat anti-human IgG Alexa fluor 488 (A-11013, Invitrogen, USA), donkey anti-rabbit IgG Alexa fluor 555 (ab150074, Abcam, UK), and goat anti-mouse IgG Alexa fluor 647 (ab1500115, Abcam, UK) were incubated for 1h at 25C. The nucleus was stained with VECTASHIELD Antifade mounting medium containing DAPI (LSbio, USA) and visualized using a Zeiss LSM 900 confocal microscope (Zeiss, German). The viral protein signals were converted to relative intensity percentages using CellProfiler 4.2.1, and the viral protein intensity was normalized to DAPI intensity.

All data were presented as the meanstandard deviation (SD). GraphPad Prism version 8 (GraphPad Software, USA) was used to analyze the data. Two-tailed Students t-test was performed to compare the means of two groups. Differences of *P<0.05, **P<0.01, ***P<0.001, or ****P<0.0001 were considered significant.

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An RNA-hydrolyzing recombinant minibody prevents both influenza A virus and coronavirus in co-infection models ... - Nature.com